Methods for cancer prognosis and diagnosis

ABSTRACT

The invention provides methods for prognosis, diagnosis, staging and disease progression in human cancer patients related to expression levels of a variety of immunohistochemical and genetic markers associated with poor cancer prognosis, and in particular those markers related to tumor invasiveness, metastasis and spread. The invention also provides methods using a predictive index for prognosis of cancer patients for metastasis, recurrence and relapse of neoplastic disease. The methods of the invention are useful for making clinical decisions on cancer treatment, surveillance and surgical intervention.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to cancer diagnosis and treatment, andspecifically to the determination of a predictive index for prognosis ofcancer patients for metastasis, recurrence and relapse of neoplasticdisease. The invention relates to the determination of a variety ofimmunohistochemical and genetic markers associated with poor cancerprognosis, and in particular those markers related to tumorinvasiveness, metastasis and spread. The invention particularly relatesto the use of certain markers associated with tumor invasiveness,metastasis and spread to provide a prognostic index for making clinicaldecisions on cancer treatment, surveillance and surgical intervention.

[0003] 2. Summary of the Related Art

[0004] Cancer remains one of the leading causes of death in the UnitedStates. Clinically, a broad variety of medical approaches, includingsurgery, radiation therapy and chemotherapeutic drug therapy arecurrently being used in the treatment of human cancer (see the textbookCANCER: Principles & Practice of Oncology, 2d Edition, De Vita et al.,eds., J. B. Lippincott Company, Philadelphia, Pa., 1985). However, it isrecognized that such approaches continue to be limited by a fundamentalinability to accurately predict the likelihood of metastasis and tumorrecurrence or the most efficacious treatment regime for minimizing theoccurrence of these negative outcomes.

[0005] The discovery and clinical validation of markers for cancer ofall types which can predict prognosis, likelihood of invasive ormetastatic spread is one of the major challenges facing oncology today.In breast cancer, for example, 70% of the approximately 186,00 annualcases present as node negative; however, 30% of these cases will recurafter local therapy (mastectomy or “lumpectomy”) (Boring et al., 1992,Clin. J. Cancer 42: 19-38). Although adjuvant chemotherapy has beendemonstrated to improve survival in node negative breast cancer patients(Mansour et al., 1989, N. Engl. J. Med. M: 485490), it remains uncertainhow to best identify patients whose risk of disease recurrence exceedstheir risk of significant therapeutic toxicity (Osbourne, 1992, J. Clin.Oncol. IQ: 679-682).

[0006] Current approaches to answer these questions stratify nodenegative breast cancer on the basis of primary tumor size, pathologicalgrade, DNA S-phase fraction (SPF) and steroid hormone receptor status(Allegra et al., 1979, Cancer Treat. Rep. 63: 1271-1277; Von Rosen etal., 1989, Breast Cancer Res. Treat. 1l: 23-32; Fischer et al., 1992, J.Natl. Cancer Inst. 11: 152-258; Clark et al., 1994, N. Engl. J. Med.320: 627-633). For example, moderately and well-differentiated tumors <1cm in size are thought to require only local excision regardless ofreceptor status, while such tumors from 1 to 3 cm in size that expressnormal levels of hormone receptor are treated with hormone therapy(Fischer et al., 1993, in Cancer Medicine, 3d ed., Holland et al., eds.,Philadelphia: Lea & Febiger, pp. 1706-1774). On the other hand, patientswith tumors larger than 2 cm that are poorly differentiated and/orhormone receptor negative are treated with adjuvant chemotherapy (1992,Lancet 339: 1-15; 1989, N. Engl. J. Med. 320: 491-496). However,therapeutic indications are much less clearly defined for patientshaving moderately differentiated tumors of 1 to 3 cm in size where thehormone receptor status is borderline or unknown (Gasparini et al.,1993, J. Natl. Cancer Inst. 85: 1206-1219). Deciding the mostappropriate therapy for this group of patients, comprising about 70,000women annually, would benefit from the development of validatedprognostic analysis. Similar prognostic tools are needed in most otherforms of cancer.

[0007] Thus, there is a need in this art for developing methods formaking clinical decisions on adjuvant therapy, tumor surveillance andthe likelihood of disease progression based on validated tumor markersstatistically correlated with tumor invasiveness, metastasis andrecurrence.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods for predicting a diseasecourse in a human cancer patient. The invention also provides aprognostic (risk) index for making predictions about disease progressionand prognosis, and for determining the proper course of treatment for anindividual patient using the index to grade the patient's tumor andestimate their chances for survival.

[0009] In a first aspect the invention provides a method for making aprognosis of disease course in a human cancer patient. The methodcomprises the following steps. First, a sample of a tumor from the humancancer patient is obtained. Then, the levels of three tumor markers inthe tumor sample are determined, and compared with levels of thesemarkers in a control, non-invasive, non-metastatic tumor sample of thesame type. The tumor markers tested are nuclear localization of p53protein (which is used as an indicator of p53 mutation), thrombospondin1 expression, and the extent of microvascularization in the tumor sample(as a measure of angiogenesis in the sample). In the practice of theinvention, a poor prognosis, that is, a prognosis of the likelihood offurther neoplastic, particularly metastatic, disease, is made when thelevel of nuclear localization of p53 in the tumor sample is greater thanthe level of nuclear localization of p53 protein in the non-invasive,non-metastatic tumor sample; the level of thrombospondin 1 expression inthe tumor sample is less than the level of thrombospondin 1 expressionin the non-invasive, non-metastatic tumor sample; and the extent ofmicrovascularization in the tumor sample is greater than the extent ofmicrovascularization in the non-invasive, non-metastatic tumor sample.

[0010] In a preferred embodiment, the determination of a poor prognosisis made when the level of nuclear localization in the tumor sample isfrom about twofold to about tenfold, more preferably about fivefold,greater than the level of nuclear localization of p53 protein in thenon-invasive, non-metastatic tumor sample.

[0011] In a preferred embodiment, the determination of a poor prognosisis made when the level of thrombospondin 1 expression in the tumorsample is from about twofold to about tenfold, more preferably aboutfivefold, less than the level of thrombospondin 1 expression in thenon-invasive, non-metastatic tumor sample.

[0012] In a preferred embodiment, the determination of a poor prognosisis made when the extent of microvascularization in the tumor sample isfrom about twofold to about tenfold, more preferably about sixfold,greater than the extent of microvascularization in the non-invasive,non-metastatic tumor sample.

[0013] In a more preferred embodiment, the determination of a poorprognosis is made when the level of nuclear localization of p53 in thetumor sample is from about twofold to about tenfold greater than thelevel of nuclear localization of p53 protein in the non-invasive,non-metastatic tumor sample, and the level of thrombospondin 1expression in the tumor sample is from about twofold to about tenfoldless than the level of thrombospondin I expression in the non-invasive,non-metastatic tumor sample and the extent of microvascularization inthe tumor sample is from about twofold to about tenfold greater than theextent of microvascularization in the non-invasive, non-metastatic tumorsample. Most preferably, the level of nuclear localization of in thetumor sample is from about fivefold greater than the level of nuclearlocalization of p53 protein in the non-invasive, non-metastatic tumorsample, the level of thrombospondin 1 expression in the tumor sample isfrom about fivefold less than the level of thrombospondin 1 expressionin the non-invasive, non-metastatic tumor sample and the extent ofmicrovascularization in the tumor sample is from about sixfold greaterthan the extent of microvascularization in the non-invasive,non-metastatic tumor sample in determining a poor prognosis for a cancerpatient.

[0014] A In preferred embodiments, the levels of nuclear localization ofp53, thrombospondin 1 expression and the extent of microvascularizationare determined by immunohistochemical staining and detected bymicroscopy.

[0015] The invention also provides methods wherein the results of thedetermination of the levels of nuclear localization of p53,thrombospondin 1 expression, and the extent of microvascularization areused to prepare a prognostic or “risk” index for making a prognosticdetermination. In this aspect of the invention, a prognostic index isprepared comprising the product of the percentage of cells in the tumorsample that are positive for nuclear localization of p53 protein and oneplus the intensity of immunohistochemical staining; the product of thepercentage of cells in the tumor sample that are positive formicrovascularization and one plus the intensity of immunohistochemicalstaining; and the product of the percentage of cells in the tumor samplethat are positive for thrombospondin 1 expression and one plus theintensity of immunohistochemical staining. In calculating theseproducts, the intensity of staining is assigned a value of 0 forstaining equal to a negative control, a value of 1 for weak staininggreater than the negative control, a value of 2 for moderate stainingintensity, a value of 3 for staining intensity equal to a positivecontrol, and a value of 4 for staining intensity greater than thepositive control. The calculated products of each of the tumor markerdeterminations are then weighted on a scale of from +1 to −4, and theindex is produced as the sum of the weighted products for nuclearlocalization of p53, thrombospondin 1 expression andmicrovascularization. In the practice of the invention, a prognosis of alikelihood of further neoplastic, particularly metastatic, disease ismade when this sum is less than about −5.

[0016] In additional embodiments, the prognostic index is produced bypreparing a weighted scale of expression levels of the tumor markersrelated to progression observed in a representative sample of aparticular tumor type, wherein the different values in the weightedscale are related to increased invasiveness or metastatic spread in therepresentative sample.

[0017] The methods of the invention are also provided for identifying ahuman cancer patient at risk for additional neoplastic disease, forstaging malignant disease in a human cancer patient and assessing therelative risk of metastatic disease versus the risk of toxicity (such asleukocytopenia, for example) from chemotherapeutic treatment.

[0018] The methods of the invention are provided for prognosis ofdisease course in a cancer patient suffering from any specific cancer ofany tissue of origin. In preferred embodiments, the cancer is breastcancer, prostate cancer or melanoma.

[0019] Specific preferred embodiments of the present invention willbecome evident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIGS. 1A through 1C are histograms of the intensity of tumormarker staining versus tumor histology for four histological subsetsdescribed in Example 1. The p values indicate significance of theobserved differences between samples of different histologies,determined using paired, one-tail t-test analysis.

[0021]FIG. 2 is a graph showing the increase in p53 nuclear accumulationand microvascularization and decrease in TSP-1 expression with tumorprogression for a cohort of breast cancer samples as described inExample 1.

[0022]FIG. 3 is a graph of a retrospective study of patient survival of40 breast cancer patients as described in Example 2, comparing patientshaving a prognostic risk index of greater than or equal to −5 (GE-5)with patients having a prognostic risk index of less than −6 (LE-6).

[0023]FIG. 4 is a graph of a retrospective study patient survival of 104prostate cancer patients as described in Example 3, comparing patientshaving a prognostic risk index of greater than or equal to −7 (GE-7)with patients having a prognostic risk index of less than −8 (LE-8).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention provides a method for making a prognosisabout disease course in a human cancer patient. For the purposes of thisinvention, the term “prognosis” is intended to encompass predictions andlikelihood analysis of disease progression, particularly tumorrecurrence, metastatic spread and disease relapse. The prognosticmethods of the invention are intended to be used clinically in makingdecisions concerning treatment modalities, including therapeuticintervention, diagnostic criteria such as disease staging, and diseasemonitoring and surveillance for metastasis or recurrence of neoplasticdisease.

[0025] The methods of the invention are preferably performed using humancancer patient tumor samples, most preferably samples preserved, forexample in paraffin, and prepared for histological andimmunohistochemical analysis.

[0026] The invention also provides an index for use with the methods ofthe invention to relate three tumor markers (p53 nuclear accumulation,thrombospondin-l expression and microvascularization) with diseaseprogression, particularly invasiveness and metastatic spread. Theindices of the invention can be prepared as described herein for anytumor type, provided that there is available a representative cohort ofsamples of the tumor type having varying degrees of tumor invasivenessand metastatic spread, to enable the production of a weighted scale ofexpression levels of the three tumor markers. Preferably, the size ofthe cohort is sufficiently large to enable statistical analyses toverify the significance of differences in tumor marker expression fordisease progression.

[0027] Indices as provided by the invention can also be constructedusing any relevant tumor marker associated with disease progression,again provided that there is available a representative cohort ofsamples of the tumor type having varying degrees of tumor invasivenessand metastatic spread, to enable the production of a weighted scale ofexpression levels of the three tumor markers. Preferably, the size ofthe cohort is sufficiently large to enable statistical analyses toverify the significance of differences in tumor marker expression fordisease progression. Additional tumor markers can be added to the threetumor markers used in the practice of this invention, or other tumormarkers can replace any of the markers described herein, if such markersmeet the proviso discussed above.

[0028] The methods of the invention are practiced by determiningexpression levels of the three preferred tumor markers (p53 nuclearaccumulation, thrombospondin-I expression and microvascularization) in ahuman cancer patient sample. In preferred embodiments, expression levelsare determine immunohistochemical. However, expression levels can bedetermined using any appropriate and convenient method. For example, insitu polymerase chain reaction (CITE) and in situ nucleic acidhybridization methods for determining expression levels of TSP-1 fallwithin the methods of the invention. Additionally, site-specificmutation analysis, including sequence analysis or mutant allele-specificamplification of mutant p53, can be used for determining expressionlevels of mutant p53 in a tumor sample. Similarly, any method fordetecting microvascularization, including any method of specificstaining, fall within the ambit of the methods of the present invention.Detection methods are chosen appropriate for the labeling oridentification of any of the three tumor markers used in the practice ofthe invention.

[0029] In a preferred embodiment, the present invention usesimmunohistochemical methods for detecting expression levels of the tumormarkers of the invention. In the practice of the invention, antibodiesor antisera, preferably polyclonal antisera, and most preferablymonoclonal antibodies specific for each marker are used to detectexpression levels, using anti-p53, anti-TSP-1 and anti-CD31 antibodyimmunostaining. Detection of these antibodies can be realized by directlabeling of the antibodies themselves, with labels including aradioactive label such as ³H, ¹⁴C, ³⁵S, ¹²⁵I or ¹³¹I, a fluorescentlabel, a hapten label such as biotin, or an enzyme such as horse radishperoxidase or alkaline phosphatase. Alternatively, unlabeled primaryantibody is used in conjunction with labeled secondary antibody,comprising antisera, polyclonal antisera or a monoclonal antibodyspecific for the primary antibody. In a preferred embodiment, theprimary antibody or antisera is unlabeled, the secondary antisera orantibody is conjugated with biotin and enzyme-linked streptavidin isused to produce visible staining for histochemical analysis.

[0030] Detection and quantitation of the tumor markers is provided usingmethods appropriate for the staining or other detection method used. Inpreferred embodiments, immunohistochemically stained sections of a tumorsample are analyzed microscopically, most preferably by light microscopyof a sample stained with a stain that is detected in the visiblespectrum, using any of a variety of such staining methods and reagentsknown to those with skill in the art. Most preferably the methods of theinvention are practiced by those with skill in the histological arts,but embodiments of the invention provided to permit relatively unskilledtechnicians to properly interpret tumor marker results are also withinthe scope of the methods provided.

[0031] The following Examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.

EXAMPLE 1 Tumor Progression/Prognosis Analysis for Breast Cancer

[0032] Nuclear localization of p53 protein, thrombospondin 1 expressionlevels and extent of microvascularization were determinedimmunohistochemically as follows.

[0033] Tumor blocks from breast cancer patients were obtained fromWestern Medical Center and H. Lee Moffitt Cancer Center and examinedindependently by two pathologists to confirm the diagnosis for tumortype and stage. Representative section of each tumor sample were chosenon the basis of pathological examination for immunohistochemicalstaining. Tissue sections 5 microns in thickness were cut and preparedon slides using standard histological preparation techniques. Sinceparaffin sections were used, slides were first deparaffinized usingHistoclear (Biogenics, Calif.). Antigens were exposed forimmunohistochemical staining by pronase digestion (for CD31 detection)and by microwave boiling (for p53 and thrombospondin 1 (TSP-1)detection) using antigen recovery solution (Biogenics). Slides were thenincubated in a solution of 3% hydrogen peroxide in distilled water atroom temperature for 10 min, then rinsed briefly with water. Slides werethen incubated for 10 min at room temperature using 100 gL goat serum asblocking buffer. Excess blocking buffer was removed from the slides byshaking, and the slides then incubated with primary antibody at roomtemperature for 30 min. The primary antibodies used in these assayswere: antibody DO1 for p53 (obtained from Santa Cruz Biotech, SantaCruz, Calif.); antibody clone 12 for TSP-1 (Immunotech, Inc., Westbrook,ME); and an endothelial cell-specific antibody reactive with the cellsurface antigen CD31 for microvascularization (Dako, Carpenteria,Calif.). Slides were rinsed twice with phosphate buffered saline (PBS)for 5 min after primary antibody incubation.

[0034] For detection of primary antibody binding, tissue sections werethen incubated with biotinylated goat antimouse immunoglobulin for 20min at room temperature in a humidified chamber (70-100% relativehumidity). Slides were rinsed twice with PBS after this incubation, andthen treated with a solution of peroxidase-conjugated streptavidin for20 at room temperature. After being rinsed again with PBS, the slideswere incubated in a solution of 3,3′-diaminobenzidine for 3 min at roomtemperature. Slides were rinsed with PBS for 5 min, exposed tohematoxylin for 1 min, rinsed with water for 10 min, dehydrated in anascending ethanol series, cleared with xylene, mounted and viewed bylight microscopy.

[0035] Microscopic analyses were performed at 200× magnification asfollows. The malignant cells on the slide were counted, and the numberof stained cells and staining intensity determined. Each slide wasscored independently by two pathologists. Scoring of staining intensitywas relative to the following scale:

[0036] 0=staining intensity equal to the negative control

[0037] 1=staining intensity weak but greater than negative control

[0038] 2=staining intensity moderate (more than negative control, butless than positive control)

[0039] 3=staining intensity strong, equal to positive control

[0040] 4=staining intensity greater than positive control.

[0041] Control slides used for comparison were paraffin-embedded MCF-740F cells (ATCC #HTB-22) for p53 and TSP-1. Microvascularizationcontrols were paraffin-embedded tumor specimens showing high reactivitywith anti-CD31 antibody.

[0042] Alternatively, TSP-1 expression was determined using imageanalysis (IA) techniques. Slides immunohistochemically stained fordetection of TSP-1 expression as described above were analyzed using aCAS 200 image analysis system (Cell Analysis Systems, Lombard, Ill.) toquantitate the staining intensity of TSP-1 marker positive cells asdescribed (see Figge et al., 1991, Amer. J. Pathol. 139: 1213-1221 andEsteban et al., 1993, Amer. J. Clin. Pathol. 22: 32-38). This analyticalmethod uses a two-color system to sample image data using 2 solid-statevideo cameras, each with its own optical filter, mounted on a lightmicroscope. Video signals are sent to an image capture board, whichsamples and digitizes the analog signal. The digital value of the signalsample is proportional to the amplitude of the video signal and isstored in an interactive computer. Measurements are obtained fromcalibrated conversion of pixel information from the video image.

[0043] For IA of TSP-1 expression in breast cancer tumor samples, theinstrument was set at threshold values optimized to distinguish betweencell membrane, nuclear and cytosolic portions of the stained image, andthe zero pixel set-point adjusted using a tumor section stained with anisotype-matched irrelevant (i.e., unrelated) antibody. At least 10fields of positive area on the slide were scanned for each tumorspecimen. Video values were converted to the product of the positiveareas and positively-stained areas, expressed as optical density (O.D.)Units using instrument software. Antigen preservation control wasevaluated using vimentin staining (1:200 antibody dilution, obtainedfrom Dako, Carpenteria, Calif.). The results of IA were consistent withresults obtained by visual analysis of the stained tumor sections.

[0044] The values of staining intensity related to “positive” or“negative” predicted outcomes were determined based on univariateanalysis of the markers on survival, using a training subset (n=42) forwhich survival data were known. Immunohistochemistry (IHC) scores wereassigned based on the product of the percentage of cells positive in thesample times (1+ intensity of staining), using the staining intensityscale described above. Tissue sections with immunodetectably nuclear p53observed in more than 5% of the cells with 2+ staining intensity(corresponding to an IHC value >15) were considered positive. (It isnoted that the presence of nuclear-located p53 is used as a marker formutant p53, consistent with the difference in cellular location ofmutant p53 versus wildtype known in the prior art (Hall & Lane, 1994, J.Pathol. 172: 1-4). For IA of TSP-1 expression, positive sections weredetermined to have a value of >30 O.D. For angiogenesis, microvesselswere counted in the region of greatest vessel density over at least 10fields; samples designated as positive had >70 vessels per field.Statistics, including Fischer's exact test and unpaired one-tailed ttest were performed using a software program (GraphPad Software, v2.05,San Diego, Calif.) to compare values for each markers' incidence andintensity of expression as a function of histological progression.

[0045] The results of these assays are shown in Tables I and II. Table Ipresents the results for the tested markers based on a dichotomy ofinvasive versus non-invasive ductal breast carcinoma (as determined bypathological examination of breast tumor samples and registryinformation provided for each sample), while Table II shows thedifference in staining patterns observed for the 4 histological subsetsstudied. These results show that highly significant changes in all threeof the tested markers were observed in the transition from non-invasiveto invasive disease. The frequency of nuclear p53 localization andmicrovascularization were found to be increased (>5-fold) significantly(p <0.0001) in invasive tumor tissue, while the frequency ofthrombospondin 1 expression decreased (>5-fold) significantly (p<0.0001)in these tumor samples.

[0046] When the tumor samples are further distinguished based on four(rather than two) subsets of morphological and histochemical criteria,additional differences were detected. As shown in Table II, frequency ofnuclear localization of p53 increased significantly (p=0.006) in acomparison between low-grade and high-grade ductal carcinoma in situ(DCIS), even though both subsets are non-invasive. In these assays,nuclear p53 staining was not detected in any of the low-grade DCISsamples, while 31% (6/22) of the high-grade DCIS samples showed positivestaining.

[0047] For the transition between high-grade (but non-invasive) DCIS tofrankly invasive ductal carcinoma with negative lymph nodes, only thedecline in TSP-1 expression was significant (p <0.002), with thefrequency of TSP-1 expression declining from 82% (18/22 samples) to 32%(6/19 samples). In addition, the transition from invasive ductalcarcinoma without lymph node metastasis to invasive disease accompaniedby lymph node metastasis showed significant changes in p53 nuclearlocalization, TSP-1 expression and microvascularization. The incidenceof samples with p53 nuclear localization in tumor samples comprisingmetastatic cancer increased from 47% (9/19 samples) to 82% (14/17samples) (p=0.041), the incidence of samples with high microvesselcounts increased from 53% (10/19 samples) to 100% (17/17 samples)(p=0.001), and the incidence of samples with pronounced TSP-1 stainingdecreased from 32% (6/19 samples) in tumor without lymph nodeinvolvement to 0% (0/17 samples) in tumors associated withmetastasis-positive lymph nodes (p=0.02). TABLE I Marker Profile:Invasive versus Non-invasive Ductal Breast Carcinoma Percent PositiveMarkers Studied: Tumor Type p53 TSP-1 Microvasc. Non-invasive (n = 48)12 83 12 Invasive (n = 36) 64 17 75 P value* <0.0001 <0.0001 <0.0001

[0048] TABLE II Marker Profile of Breast Carcinoma Progression PercentPositive Markers Studied: Tumor Type p53 TSP-1 Microvasc. Low-gradeDCIS¹ (n = 26)  0 89  4 High-grade DCIS (n = 36)  31* 82 23 Invasive -LN(−)² (n = 19) 47  32* 53 Invasive - LN(+)³ (n = 17)  82*  0 100*

[0049] These results demonstrated that nuclear localization of p53,decreased thrombospondin 1 expression, and increasedmicrovascularization were significantly correlated with increasedinvasiveness of primary breast cancer, increased metastasis, and poorerprognosis for breast cancer patients whose tumors had these markers. Todetermine whether disease progression was linked not only to theincidence, but also the degree of marker expression as well, theintensity of staining of the markers as determined above byimmunohistochemistry or image analysis was plotted versus tumorhistology for the four histological subsets described above. Theseresults are shown in FIGS. 1A through 1C. These results demonstrate adistinct pattern of differences in intensity and degree of expression ofthe three tumor markers assayed above. These results show that nuclearp53 accumulation and the number of tumor microvessels increased in boththe transition from low-grade DCIS to high-grade DCIS, and also in thetransition from invasive tumors without evidence of metastatic spread toinvasive tumors having metastasis-positive lymph node involvement FIGS.1A and 1B). TSP-1 expression showed a significant decline in intensitybetween high-grade DCIS and invasive cancer prior to metastatic spread.

[0050] These results are also shown graphically in FIG. 2, where theincrease in p53 nuclear accumulation and microvascularization anddecrease in TSP-1 expression with tumor progression is shown. These datasuggest that a coordinated relationship existed between nuclear p53accumulation and angiogenesis, while TSP-1 expression was inverselycorrelated with these two factors. Invasion and metastasis in breastcancer were associated in a statistically-significant way withacquisition of dysfunctional p53 (as evidenced by nuclear accumulation),decreased TSP-1 expression, and increased angiogenesis.

EXAMPLE 2 Tumor Prognostic Index

[0051] The results obtained in the assays described in Example 1 abovewere used to construct a prognostic (risk) index relating tumorprogression and increasingly poorer disease prognosis with positivemarker results, graded by intensity of immunohistochemical staining ofeach of the tumor markers.

[0052] The IHC scores obtained in Example 1 were used to construct atumor progression/prognosis (risk) index as follows. Scores for each ofthe markers were associated with a integer index scale from +1 to −4.This index scale was constructed for each marker based on the followingIHC staining results as follows. The p53 nuclear accumulation score wasderived from the percentage of cell staining with anti-p53 antibodymultiplied by (1 +intensity of staining), using the intensity ofstaining and positive control cells described in Example 1. The TSP-1score was derived from IA data obtained as described in Example 1, asweighted based on the percentage of cells staining positively for TSP-1.Angiogenesis was scored as greatest number of microvessels per fieldstained with anti CD31 antibody, after a minimum scan of 10 fields.These scores and their associated weighted index scores are describedbelow in Table III. TABLE III p53, TSP-1 and Angiogenesis Indices Indexp53 Score Thrombospondin Score Microvascularization Score 1  0-30 >30 0-30 0 31-60 25-29 31-70 −1 61-90 20-24 71-85 −2 91-120 15-19  86-100−3 121-450 10-14 101-123 −4 >150 0-9 >123

[0053] The tumor prognosis (risk) index is then prepared by the sum ofthe index scores for p53 accumulation, TSP-1 expression and angiogenesis(microvascularization), with poor prognosis being determined for tumorshaving an summed index score of −5 or less.

[0054] The efficacy the prognostic (risk) index was assessed using Logrank tests on survival versus index score. The significance of the indexscores on survival are shown in Table IV. In this table, it can be seenthat a statistically-significant difference was observed in survivalbetween patients having tumors with a summed index score ≧−5 whencompared with patients having tumors with a summed index score ≦−6.Similarly, a statistically-significant difference was observed insurvival between patients having tumors with a summed index score ≧⊕6when compared with patients having tumors with a summed index score <−7.Finally, there was a statistically-significant difference was observedin survival between patients having tumors with a summed index score >−7when compared with patients having tumors with a summed index score <−8.TABLE IV Log Rank Tests to Compare Survival Index Score Log Rank Test PValue ≧−5 vs. <−6 p < 0.0001 ≧−6 vs. <−7 p < 0.0007 ≧−7 vs. <−8 p <0.0548

[0055] These results are shown graphically in FIG. 3 in a retrospectiveanalysis of 40 breast cancer patients having a summed prognostic (risk)index score greater than −5 (GE −5) or less than −6 (LE −6). In thisstudy, survival was correlated with risk index scores greater than orequal to −5 (about 80% survival at 60 months post-diagnosis) versusabout 20% survival for patient having tumors with index scores less than−6.

[0056] These results were also analyzed using multivariate analysisincluding angiogenesis, tumor size and lymph node status. These resultsshowed that the index was more predictive of patients' prognosis forsurvival than the commonly-used indices of tumor size or lymph nodestatus.

[0057] These results demonstrate that the tumor progression index is astatistically reliable predictor of tumor prognosis and diseaseprogression for breast cancer.

EXAMPLE 3 Tumor Progression/Prognosis Analysis for Prostate Cancer

[0058] The immunohistochemical analyses described above in Example 1were applied to prostate cancer samples. In addition, androgen receptorexpression was assayed for these tumors, due to the recognizedcorrelation between androgen receptor expression and poorprognosis/survival in these patients.

[0059] In this study, 104 prostate cancer patient tumor samples wereassayed for nuclear accumulation of p53, TSP-1 expression, androgenreceptor (AR) gene expression and microvascularization. These assayswere performed immunohistochemically as described above in Example 1,except that androgen receptor expression was determined using an anti-ARantibody (Biogenics, used at 1:20 dilutions).

[0060] The results of these studies closely paralleled the resultsobtained with breast carcinoma, and the indices derived from the p53,TSP-1 and angiogenesis/microvascularization data were identical to thoseshown in Table III. In addition, AR expression was found to benegatively correlated with prognosis and survival using multivariateanalysis (Cox regression analysis), which showed statisticalsignificance (p=0.0077). Interestingly, the presence of nuclearaccumulation of p53 was correlated with AR expression (p<0.0041).

[0061] The risk index incorporating levels of nuclear p53, TSP-1expression and angiogenesis was found to be significantly associatedwith survival. Survival in patients with a risk index of −8 or less wassignificantly lower than that in patients with a prognostic (risk) indexof −7 or greater (p<0.0001). The risk index was also associated withsurvival (p<0.0061) even after adjustment for age and stage.

[0062] These results are shown graphically in FIG. 4. This Figureillustrates the results of a retrospective analysis of 104 prostatecancer patients having a summed prognostic (risk) index score greaterthan −7 (GE −7) or less than −8 (LE −8). In this study, survival wascorrelated with risk index scores greater than or equal to −7 (about 95months to 20% survival post-diagnosis) versus about 30 months to 20%survival post-diagnosis for patients having tumors with index scoresless than −8. These results demonstrated that the prognosis (risk) indexwas reliable for predicting poor prognosis/increased disease progressionbased on the tested tumor markers.

[0063] It should be understood that the foregoing disclosure emphasizescertain specific embodiments of the invention and that all modificationsor alternatives equivalent thereto are within the spirit and scope ofthe invention as set forth in the appended claims.

We claim:
 1. A method for making a prognosis of disease course in ahuman cancer patient, the method comprising the steps of: (a) obtaininga sample of a tumor from the human cancer patient; (b) determining alevel of nuclear localization of p53 protein in the tumor sample andcomparing the level of nuclear localization of p53 protein in the tumorsample with the level of nuclear localization of p53 protein in anon-invasive, non-metastatic tumor sample; (c) determining a level ofthrombospondin 1 expression in the tumor sample and 4910 comparing thelevel of thrombospondin 1 expression in the tumor sample with the levelof thrombospondin 1 expression in a non-invasive, non-metastatic tumorsample; (d) determining by immunohistochemistry an extent ofmicrovascularization in the tumor sample and comparing the extent ofmicrovascularization in the tumor sample with the extent ofmicrovascularization in a non-invasive, non-metastatic tumor sample; and(e) preparing a prognostic index comprising the results of thedetermination of the levels of nuclear localization of p53,thrombospondin 1 expression, and the extent of microvascularization inthe tumor sample, wherein said prognosis is predicted from considering alikelihood of further neoplastic disease which is made when the level ofnuclear localization of in the tumor sample is greater than the level ofnuclear localization of p53 protein in the non-invasive, non-metastatictumor sample; the level of thrombospondin 1 expression in the tumorsample is less than the level of thrombospondin 1 expression in thenon-invasive, non-metastatic tumor sample; and the extent ofmicrovascularization in the tumor sample is greater than the extent ofmicrovascularization in the non-invasive, non-metastatic tumor sample.2. The method of claim 1, wherein the level of nuclear localization ofp53 protein in the tumor sample is from about twofold to about tenfoldgreater than the level of nuclear localization of p53 protein in thenon-invasive, non-metastatic tumor sample.
 3. The method of claim 1,wherein the level of thrombospondin 1 expression in the tumor sample isfrom about twofold to about tenfold less than the level ofthrombospondin 1 expression in the non-invasive, non-metastatic tumorsample.
 4. The method of claim 1, wherein the extent ofmicrovascularization in the tumor sample is from about twofold to abouttenfold greater than the extent of microvascularization in thenon-invasive, non-metastatic tumor sample.
 5. The method of claim 1,wherein the level of nuclear localization of p53 protein in the tumorsample is from about twofold to about tenfold greater than the level ofnuclear localization of p53 protein in the non-invasive, non-metastatictumor sample, and wherein the level of thrombospondin 1 expression inthe tumor sample is from about twofold to about tenfold less than thelevel of thrombospondin 1 expression in the non-invasive, non-metastatictumor sample and wherein the extent of microvascularization in the tumorsample is from about twofold to about tenfold greater than the extent ofmicrovascularization in the non-invasive, non-metastatic tumor sample.6. The method of claim 1, wherein the level of nuclear localization ofp53 protein in the tumor sample is from about fivefold greater than thelevel of nuclear localization of p53 protein in the non-invasive,non-metastatic tumor sample, and wherein the level of thrombospondin 1expression in the tumor sample is from about fivefold less than thelevel of thrombospondin 1 expression in the non-invasive, non-metastatictumor sample and wherein the extent of microvascularization in the tumorsample is from about sixfold greater than the extent ofmicrovascularization in the non-invasive, non-metastatic tumor sample.7. The method of claim 1, wherein the level of nuclear localization ofp53, the level of thrombospondin 1 expression and the extent ofmicrovascularization are determined by immunohistochemical staining. 8.The method of claim 1 wherein the cancer is breast cancer.
 9. The methodof claim 1 wherein the cancer is prostate cancer.
 10. The method ofclaim 1 wherein the cancer is melanoma.
 11. A method for making aprognosis of disease course in a human cancer patient, the methodcomprising the steps of: (a) obtaining a sample of a tumor from thehuman cancer patient; (b) determining a level of nuclear localization ofp53 protein in the tumor sample and comparing the level of nuclearlocalization of p53 protein in the tumor sample with the level ofnuclear localization of p53 protein in a non-invasive, non-metastatictumor sample; (c) determining a level of thrombospondin 1 expression inthe tumor sample and comparing the level of thrombospondin 1 expressionin the tumor sample with the level of thrombospondin 1 expression in anon-invasive, non-metastatic tumor sample; (d) determining byimmunohistochemistry an extent of microvascularization in the tumorsample and comparing the extent of microvascularization in the tumorsample with the extent of microvascularization in a non-invasive,non-metastatic tumor sample; and (e) preparing an index comprising (I)the product of the percentage of cells in the tumor sample that arepositive for nuclear localization of p53 protein multiplied by the sumof (one plus the intensity of immunohistochemical staining); (ii) theproduct of the percentage of cells in the tumor sample that are postivefor microvascularization multiplied by the sum of (one plus theintensity of immunohistochemical staining); and (iii) the product of thepercentage of cells in the tumor sample that are positive forthrombospondin 1 expression multiplied by the sum of (one plus theintensity of immunohistochemical staining); wherein for steps (e)(I) and(e)(ii) the intensity of staining is assigned a value of 0 for stainingequal to a negative control, a value of 1 for weak staining greater thanthe negative control, a value of 2 for moderate staining intensity, avalue of 3 for staining intensity equal to a positive control, and avalue of 4 for staining intensity greater than the positive control, andwherein for step (e)(iii) the intensity of staining is assigned a valueof 4 for staining equal to or greater than a negative control, a valueof 3 for staining slightly decreased from the negative control, a valueof 2 for staining intensity moderately decreased from the negativecontrol, a value of 1 for staining intensity equal to a positivecontrol, and a value of 0 for staining intensity less than the positivecontrol, and a value of 0 for staining less than the positive control;wherein the products of steps (e)(I), (e)(ii) and (e)(iii) are weightedon a scale from +1 to −4 and wherein the index comprises the sum of theweighted products for nuclear localization of p53, thrombospondin 1expression and microvascularization, wherein a prognosis of a likelihoodof further neoplastic disease is made when said sum is −5 or less,wherein said prognosis is predicted from considering a likelihood offurther neoplastic disease which is made when the level of nuclearlocalization of in the tumor sample is greater than the level of nuclearlocalization of p53 protein in the non-invasive, non-metastatic tumorsample; the level of thrombospondin 1 expression in the tumor sample isless than the level of thrombospondin 1 expression in the non-invasive,non-metastatic tumor sample; and the extent of microvascularization inthe tumor sample is greater than the extent of microvascularization inthe non-invasive, non-metastatic tumor sample.
 12. The method of claim11 wherein the index has a value of −5, −6, −7 or −8.
 13. The method ofclaim 11 wherein the cancer is breast cancer.
 14. The method of claim 11wherein the cancer is prostate cancer.
 15. The method of claim 11wherein the cancer is melanoma.
 16. The method of claim 1, wherein theprognosis of disease course includes a risk for metastasis, recurrenceand relapse of neoplastic disease.
 17. The method of claim 1, whereinthe prognosis of disease course includes staging malignant disease in ahuman cancer patient.
 18. The method of claim 11, wherein the prognosisof disease course includes a risk for metastasis, recurrence and relapseof neoplastic disease.
 19. The method of claim 11, wherein the prognosisof disease course includes staging malignant disease in a human cancerpatient.
 20. The method of claim 1, wherein the prognostic index isproduced by preparing a weighted scale of expression levels of the tumormarkers related to progression observed in a representative sample of aparticular tumor type, wherein the different values in the weightedscale are related to increased invasiveness or metastatic spread in therepresentative sample.